Journal: bioRxiv

Article Title: Actomyosin contractility modulates Wnt signaling through adherens junction stability

doi: 10.1101/220178

Figure Lengend Snippet: (A,B) Wnt-responsive luciferase TOPFLASH assay measuring TCF/LEF reporter activity in (A) MCF7 and (B) RKO cells following Wnt3A transfection and siPP1β and siMYPT3 transfection. Data are presented as mean ± SEM with letters above representing significant difference from corresponding column, (P<0.01). (C) Western blot analysis of total cellular levels of E-cad, β-cat, MYPT3, Wnt3A and PP1β. β-tub was used as a loading control. (D) Western blot analysis of β-cat in cytoplasmic (Cyto), membranous (Mem), chromatin-bound (CB), and whole cell extract (WCE) fractions in MCF7 cells. GAPDH, Na + /K + ATPase, and Histone 3 were used as loading controls for corresponding fractions. Complete fraction, see . (E) Quantification of relative levels of β-cat from each fraction taken from (D), normalized to Control + siScramble conditions. Data shown as mean ± SEM (n = 4 biological replicates), ns = not significant, *P<0.05.

Article Snippet: Proteins were transferred onto nitrocellulose membranes, and probed against the following primary antibodies: rabbit anti-β-catenin (1:1000, Cell Signaling), rabbit anti-E-cadherin (1:1000, Cell Signaling), mouse anti-PPP1R16A (MYPT-3) (1:500, abcam), mouse anti-Protein Phosphatase 1 beta (PP1β) (1:1000, abcam), rabbit anti-Wnt3A (1:1000, Cell Signaling), mouse anti-β-tubulin (1:1000, ABM), rabbit anti-GAPDH (1:3000, Cell Signaling), mouse anti-Na+/K+ ATPase (1:50 DSHB), rabbit anti-Histone H3 (1:1000 Cell Signaling).

Techniques: Luciferase, TOPFlash assay, Activity Assay, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: Active EB1 surges promote tubulin influx into the growing outer segments of the bipartite olfactory cilia in Drosophila

doi: 10.1101/2024.09.10.612170

Figure Lengend Snippet: A) Copurification of EB1:GFP from the chaGal4>EB1:GFP expressing Drosophila head extracts with the recombinant Glutathione S-transferase (GST)-tagged, KLP68D tail (GST-KLP68DT) fragment using affinity chromatography. The arrow indicates the EB1:GFP band. B) The immune-coprecipitation (IP) of the recombinant KLP68D from the head extracts of chaGal4>Klp68D:YFP and chaGal4>Klp68D(ΔT)YFP, expressing UAS-EB1FLAG using anti-FLAG. The arrow indicates a full-length KLP68DYFP band.

Article Snippet: Proteins from these gels were transferred onto a previously activated PVDF membrane (Hybond-P, GE Healthcare Ltd) in an electro-blotting apparatus (Bio-Rad, USA) following the supplier’s protocol and incubated in different primary antisera solutions as the following: anti-GFP Rabbit (dilution, 1:500; #3999 Bio Vision Inc., CA, USA) or anti-FLAG Rabbit (1:500; #F7425 Sigma-Aldrich, USA) Or anti-GST mouse (1:1000, Bioklone Biotech Pvt.

Techniques: Copurification, Expressing, Recombinant, Affinity Chromatography

Journal: bioRxiv

Article Title: Actomyosin contractility modulates Wnt signaling through adherens junction stability

doi: 10.1101/220178

Figure Lengend Snippet: (A) Salivary glands from control or dpp>flw-RNAi stained for fz3 expression. (B) Localization of Dsh-GFP and FLAG-Axin in the proximal cells of the salivary gland, identified in the dashed line area of (A), (C-C”) Effects of flw-RNAi on ectopic Dll-lacZ in wing imaginal discs: (C) RFP marked flip-out clones expressing Fz-Arr and GFP or flw-RNAi (C) GFP-positive axin nul1 MARCM clones and flw-RNAi in axin nul1 MARCM clones. (C”) Arm S10 flip out clones with GFP or flw-RNAi. (D,D’) Effects of flw-RNAi on Arm distribution in GFP-marked axin null cells. (D) DAPI was used to identify nuclei, and F-actin to mark the edges of the cell. (D’) Percent of nuclear Arm in cells was measured as an intensity plot (dotted line D) in wild type (n = 16), axin nul1 (n = 20), and axin null , flw-RNAi cells (n = 15). Data presented as mean ± SEM; ***p< 0.001. Scale bars: (A) 100 μm, (B-C”) 50 μm, (D) 5 μm.

Article Snippet: The following primary antibodies and dilutions were used: mouse anti-β-galactosidase (1:2000 Promega), mouse anti-Wg (1:100 DSHB), mouse anti-Arm (1:50 DSHB), rabbit anti-cleaved Caspase 3 (1:100 Cell Signaling), guinea pig anti-Sens (1:500, a gift from Hugo Bellen, Dept. of Molecular and Human Genetics, Baylor College of Medicine, USA), mouse anti-Dll (1:300, a gift from Ian Duncan, Dept. of Biology, Washington University in St. Louis, USA), rabbit anti-Phospho-Myosin Light Chain 2 (Ser19) (p-MyoII) (1:25 Cell Signaling), mouse anti-GFP (1:500, Cell Signaling), rabbit anti-FLAG (1:200, Sigma), rat anti-DEcad (extracellular domain) (1:50 DSHB), rat anti-Ci (1:50, DSHB).

Techniques: Staining, Expressing, Clone Assay

Journal: bioRxiv

Article Title: Drosophila melanogaster Toll-9 elicits antiviral immunity against Drosophila C virus

doi: 10.1101/2024.06.19.599730

Figure Lengend Snippet: (A) In silico prediction of signal peptide in Toll-9 protein sequence. Red solid line indicates predicted n-terminal region, orange solid line indicates the predicted center hydrophobic region, and yellow solid line indicates predicted c-terminal region of signal peptide. Black dotted line indicates the cleavage site (CS) of the signal peptide. Sec/SPI: Sec translocon transported secretory signal peptide/Signal Peptidase I Tat/SPI: Tat translocon transported Tat signal peptides/Signal Peptidase I (B) Western blot analysis demonstrating the presence of Toll-9/V5 in endosomes. Endosomal fractions were identified using Rab5 as a microsomal marker, while Actin served as a cytosolic marker. Data are representative of three independent experiments. (C) Micrographs show colocalization of Rab5-early endosome marker (green) and Toll-9 (anti-V5 tag ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (D) Micrographs show colocalization of Rab7-Late endosome marker (green) and Toll-9 (anti-V5 tag ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (E) Micrographs show colocalization of Rab5-early endosome marker (green) and Poly(I:C) (J2 anti-dsRNA ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (F) Toll-9 (anti-V5 tag ab-Green) and Poly(I:C) (J2 anti-dsRNA ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (G) Western blot analysis using the indicated antibodies following immunoprecipitation of V5 tag (Toll-9) using J2 dsRNA antibody from the lysate of Poly (I:C) treated Toll-9 OE and S2 cells in presence and absence of CuSO 4 (500 µM). Data are representative from three independent experiments.

Article Snippet: The cells were blocked in phosphate-buffered saline (PBS) containing 10% FBS and incubated with antibodies against Rab5 (1:50; Abcam ab31261), Rab7(1:20; Developmental Studies Hybridoma Bank Rab7), DCV Capsid (1:200; Abcam ab92954), dsRNA J2 (1:200; Jena Biosciences RNT-SCI-10010200) and V5 tag (1:200; ThermoFisher Scientific R960-25) for overnight at 4°C.

Techniques: In Silico, Sequencing, Western Blot, Marker, Immunoprecipitation